Bottom Line:
There is a long-standing association between wound healing and cancer, with cancer often described as a "wound that does not heal".In an adult model of chronic wounding in zebrafish, we show that repeated wounding with subsequent inflammation leads to a greater incidence of local melanoma formation.We find a strong correlation between neutrophil presence at sites of melanoma ulceration and cell proliferation at these sites, which is associated with poor prognostic outcome.

fig05: Neutrophil, but not macrophage, recruitment is correlated to the presence and to the extent of ulceration in human melanomaA–C’ Typical non-ulcerated, moderate and excessively ulcerated melanomas, respectively, all immunostained for pan-cytokeratin (brown) to illustrate epidermal wound margins. (A’, B’ and C’) High magnification details from (A, B and C) (highlighted by red boxes) co-stained for CD66b to illustrate neutrophil accumulation.D–F’ Parallel sections from the same patient blocks as in (A-C’), but stained for CD163 and pan-cytokeratin to illustrate macrophage influx.G Graph showing the correlation between extent of ulceration and extent of neutrophil influx (P < 0.0001; R2 = 0.19). A, B and C indicate the patient points from whom corresponding neutrophil immunostaining data are shown.H In contrast to (G), no correlation between extent of ulceration and extent of macrophage influx was observed (P = 0.9; R2 = 0.0004). D, E and F indicate the patient points from whom corresponding macrophage immunostaining data are shown.Data information: Scale bars represent 3 mm (A and D), 6 mm (B, C, E and F) and 200 μm (A’, B’, C’, D’, E’ and F’).

Mentions:
While there is considerable anecdotal evidence for wound-exacerbated cancer progression (Hofer et al, 1998), our zebrafish studies prompted us to investigate whether this association was linked to innate immune cell influx at the wound site. It is already established that ulceration of melanoma is a bad prognostic indicator (Balch et al, 2011), and so we examined whether this might be due to wound inflammation. Haematoxylin and eosin staining of sections allows us to delineate the extent of the epidermal wound and to categorise melanomas as having no ulceration, minimal/moderate ulceration (<70% of total tumour length) or excessive ulceration (>70% of tumour length) (In ‘t Hout et al, 2012) (Fig5). Our co-staining for CD66b+ neutrophils and CD163+ macrophages and automated software for quantification of leucocyte numbers reveal a 15 times increase in neutrophils from non-ulcerated to minimal/moderate ulcerated lesions and 100 times increase from non-ulcerated to excessively ulcerated lesions (Fig5A–C and G), whereas there is no such correlation with macrophage numbers and ulceration (Fig5D–F and H, Supplementary Table S1). We observe that recruited neutrophils can be located throughout the melanoma, but are often associated with the superficial wound site in ulcerated lesions (Fig5B and C). We see also a significant correlation between tumour cell proliferation [as assessed by Ki67/MelanA double staining (Nielsen et al, 2013)] and extent of neutrophil influx (Fig6A’, B’, C’ and D). This association appears strongest only up to minimal/moderate ulcers, suggesting that melanoma may reach a proliferation plateau soon after initial ulceration when they first become infiltrated by neutrophils, although we have no means to determine at which time point this might have occurred relative to our biopsies. We see no significant correlation between macrophage numbers and tumour cell proliferation (Fig6E and Supplementary Table S2).

fig05: Neutrophil, but not macrophage, recruitment is correlated to the presence and to the extent of ulceration in human melanomaA–C’ Typical non-ulcerated, moderate and excessively ulcerated melanomas, respectively, all immunostained for pan-cytokeratin (brown) to illustrate epidermal wound margins. (A’, B’ and C’) High magnification details from (A, B and C) (highlighted by red boxes) co-stained for CD66b to illustrate neutrophil accumulation.D–F’ Parallel sections from the same patient blocks as in (A-C’), but stained for CD163 and pan-cytokeratin to illustrate macrophage influx.G Graph showing the correlation between extent of ulceration and extent of neutrophil influx (P < 0.0001; R2 = 0.19). A, B and C indicate the patient points from whom corresponding neutrophil immunostaining data are shown.H In contrast to (G), no correlation between extent of ulceration and extent of macrophage influx was observed (P = 0.9; R2 = 0.0004). D, E and F indicate the patient points from whom corresponding macrophage immunostaining data are shown.Data information: Scale bars represent 3 mm (A and D), 6 mm (B, C, E and F) and 200 μm (A’, B’, C’, D’, E’ and F’).

Mentions:
While there is considerable anecdotal evidence for wound-exacerbated cancer progression (Hofer et al, 1998), our zebrafish studies prompted us to investigate whether this association was linked to innate immune cell influx at the wound site. It is already established that ulceration of melanoma is a bad prognostic indicator (Balch et al, 2011), and so we examined whether this might be due to wound inflammation. Haematoxylin and eosin staining of sections allows us to delineate the extent of the epidermal wound and to categorise melanomas as having no ulceration, minimal/moderate ulceration (<70% of total tumour length) or excessive ulceration (>70% of tumour length) (In ‘t Hout et al, 2012) (Fig5). Our co-staining for CD66b+ neutrophils and CD163+ macrophages and automated software for quantification of leucocyte numbers reveal a 15 times increase in neutrophils from non-ulcerated to minimal/moderate ulcerated lesions and 100 times increase from non-ulcerated to excessively ulcerated lesions (Fig5A–C and G), whereas there is no such correlation with macrophage numbers and ulceration (Fig5D–F and H, Supplementary Table S1). We observe that recruited neutrophils can be located throughout the melanoma, but are often associated with the superficial wound site in ulcerated lesions (Fig5B and C). We see also a significant correlation between tumour cell proliferation [as assessed by Ki67/MelanA double staining (Nielsen et al, 2013)] and extent of neutrophil influx (Fig6A’, B’, C’ and D). This association appears strongest only up to minimal/moderate ulcers, suggesting that melanoma may reach a proliferation plateau soon after initial ulceration when they first become infiltrated by neutrophils, although we have no means to determine at which time point this might have occurred relative to our biopsies. We see no significant correlation between macrophage numbers and tumour cell proliferation (Fig6E and Supplementary Table S2).

Bottom Line:
There is a long-standing association between wound healing and cancer, with cancer often described as a "wound that does not heal".In an adult model of chronic wounding in zebrafish, we show that repeated wounding with subsequent inflammation leads to a greater incidence of local melanoma formation.We find a strong correlation between neutrophil presence at sites of melanoma ulceration and cell proliferation at these sites, which is associated with poor prognostic outcome.